The purpose of this project was to examine the release of cholesterol and phospholipid from activated human and rat platelets and to examine the chemical and structural characteristics of the released particle containing these lipids. Increasing amounts of cholesterol and phospholipid were continuously released from platelets activated with thrombin and incubated for a period up to 2 hrs. Cholesterol release from platelets stimulated by strong platelet agonists such as collagen and the calcium ionophore A23187 but not by the weak agonist ADP. Colchicine substantially inhibited cholesterol release. The cholesterol and phospholipid that was released from platelets was contained within particles that were spherical or irregular in shape with sizes ranging from 50 to 550 nm. The particles had between 20 to 240 peripheral 18-nm long, rod-shaped projections per particle extending from a bilayered membrane surrounding an inner core. Chemical analysis showed that these particles contained 8% cholesterol, 32% phospholipids, 3% triglycerides, and 57% protein. The cholesterol to phospholipid molar ratio of the particles was about 0.6 (a value typical for plasma membrane), whereas, the ratio of cholesterol to phospholipid for particles from platelets of rats fed a high-cholesterol diet was 1.0. Polyacrylamide gel electrophoresis of particle proteins in SDS-containing buffer showed a major protein component with an apparent M.W. of 42,000, presumably actin. The data suggest that the released particles are formed as a result of vesiculation of the platelet plasma membrane. Cholesterol release did not appear to result from simple lysis of platelets since the release of lactate dehydrogenase (a measure of lysis) and cholesterol could be dissociated. We have now identified platelet-derived, cholesterol-phospholipid particles that may play a role not only in coagulation by providing phospholipid but also in atherosclerosis by providing a source of cholesterol.